Method for the direct detection of methicillin-resistant staphylococcus aureus

ABSTRACT

Described is a method for the detection and quantification of methicillin-resistant  Staphylococcus aureus  from a specimen comprising a) contacting the specimen with Anti-Protein A antibodies so as to adsorb methicillinresistant  Staphylococcus aureus  and/or methicillin-sensitive  Staphylococcus aureus , b) separating said antibodies to which methicillin-resistant  Staphylococcus aureus  and/or methicillin-sensitive  Staphylococcus aureus  have been adsorbed from the specimen, c) lysing methicillin-resistant  Staphylococcus aureus  and/or methicillin-sensitive  Staphylococcus aureus  adsorbed to said antibodies so as to release their DNA, d) combining the released DNA with (i) probes and/or primers which are specific for a target DNA sequence of the mecA gene of methicillin -resistant  Staphylococcus aureus  and/or of methicillin-resistant  Staphylococcus  epidermis, (ii) probes and/or primers which are specific for a target DNA sequence of methicillin-resistant  Staphylococcus aureus  other than a target DNA sequence of the mecA gene and (iii) probes and/or primers which are specific for a target DNA sequence of methicillin-resistant  Staphylococcus  epidermis other than a target DNA sequence of the mecA gene, whereby the target DNA sequences of methicillin-resistant  Staphylococcus aureus  and of methicillin-resistant  Staphylococcus  epidermis are not homologous, e) subjecting the combined released DNA and the specific probes and/or primers to conditions which permit amplification of said target DNA sequences, f) detecting the presence and amount of the amplified target DNA sequences as an indication of the presence and amount of methicillin-resistant  Staphylococcus aureus , new primers and probes and diagnostic kits for the detection and quantification of MRSA.

[0001] The present invention relates to a novel method for the detectionand quantification of methicillin-resistant Staphylococcus aureus (MRSA)from a specimen. Furthermore, the present invention relates to newprimers and probes and diagnostic kits for the detection andquantification of MRSA.

[0002] MRSA is a pathogenic bacterium and belongs to the class ofmultiresistant bacteria. The increasing prevalence of MRSA has become amajor clinical problem. Therefore, rapid and accurate detection andidentification of MRSA in clinical specimen are considered to becritical toward the prevention and treatment of infectious diseases.

[0003] Methods for the detection and identification of MRSA have alreadybeen described. For example, DNA-based methods employing PCR andamplification primers for the species-specific detection, identificationand quantification of Staphylococci and of the antibiotic resistancegene coding for methicillin-resistance (mecA gene) has been described inWO 98/20157.

[0004] WO 99/16780 describes the use of primers specific for the mecAgene and Staphylococcus species-specific primers for the femA gene, aregulatory gene involved in cell wall metabolism, for amplificationusing multiplex PCR. Taking endotracheal aspirates as specimen,susceptible Staphylococcus aureus and methicillin resistantcoagulase-negative Staphylococci were detected by electrophoresis.

[0005] U.S. Pat. No. 5,702,895 describes a multiplex PCR method for thedetection of MRSA using biotin-labelled and dinitrophenyl-labelledprimers for mecA and the spa gene, a marker which distinguishesStaphylococcus aureus from other Staphylococci. Specimens were takenfrom cultures of isolated bacteria and amplified DNA was detected byabsorbance.

[0006] The methods described have been used only for the analysis ofspecimens taken from cultures of isolated bacteria and sterile sites.When a non-sterile specimen is used, however, the number of MRSAcontained therein may be small and further Staphylococci such asmethicillin-sensitive Staphyloccoccus aureus (MSSA) andmethicillin-sensitive Staphylococcus epidermis (MSSE) as well asmethicillin-resistant Staphylococcus epidermis (MRSE) may be present.Under such situations the methods described are disadvantageous in thatthe occurrence of the mecA gene cannot be linked explicitly andunequivocally to the presence of MRSA. Furthermore, under suchconditions the risk of non-specific amplification cannot be excludedcompletely and sensitivity of the method is decreased.

[0007] The object of the present invention is to provide an improvedmethod for the detection and quantification of MRSA from a specimenwhich allows for highly sensitive quantification of MRSA even if thespecimen is from a non-sterile site.

[0008] This object has been achieved with a novel method according toclaim 1. According to the invention methicillin-resistant Staphylococcusaureus are detected and quantified from a specimen comprising

[0009] a) contacting the specimen with Anti-Protein A antibodies so asto adsorb methicillin-resistant Staphylococcus aureus and/ormethicillin-sensitive Staphylococcus aureus,

[0010] b) separating said antibodies to which methicillin-resistantStaphylococcus aureus and/or methicillin-sensitive Staphylococcus aureushave been adsorbed from the specimen

[0011] c) lysing methicillin-resistant Staphylococcus aureus and/ormethicillin-sensitive Staphylococcus aureus adsorbed to said antibodiesso as to release their DNA,

[0012] d) combining the released DNA with (i) probes and/or primerswhich are specific for a target DNA sequence of the mecA gene ofmethicillin-resistant Staphylococcus aureus and/or ofmethicillin-resistant Staphylococcus epidermis, (ii) probes and/orprimers which are specific for a target DNA sequence ofmethicillin-resistant Staphylococcus aureus other than a target DNAsequence of the mecA gene and (iii) probes and/or primers which arespecific for a target DNA sequence of methicillin-resistantStaphylococcus epidermis other than a target DNA sequence of the mecAgene, whereby the target DNA sequences of methicillin-resistantStaphylococcus aureus and of methicillin-resistant Staphylococcusepidermis are not homologous,

[0013] e) subjecting the combined released DNA and the specific probesand/or primers to conditions which permit amplification of said targetDNA sequences,

[0014] f) detecting the presence and amount of the amplified target DNAsequences as an indication of the presence and amount ofmethicillin-resistant Staphylococcus aureus.

Specimen

[0015] Specimens from which MRSA can be detected and quantified with thepresent invention are from sterile and/or non-sterile sites. Sterilesites from which specimens can be taken are body fluids such as blood,urine, cerebrospinal fluid, synovial fluid, pleural fluid, pericardialfluid, intraocular fluid, tissue biopsies or endotracheal aspirates.Cultures of isolated bacteria are defined herein as well as sterilesites. Non-sterile sites from which specimens can be taken are e.g.sputum, stool, swabs from e.g. skin, inguinal, nasal and/or throat.Preferably, specimens from non-sterile sites, more preferably inguinaland/or nasal swabs are used in the present invention.

Contacting the Specimen with Anti-Protein A Antibodies

[0016] Usually, the specimen will be resuspended in e.g. saline, brothsolution or bacterial culture medium such as CS medium (Brain-HeartInfusion with colistin and 2.5% NaCl) before being contacted with theAnti-Protein A antibodies. Usually, passivating agents e.g. serumalbumin will be added to the resuspended specimen.

[0017] Anti-Protein A antibodies used in the present invention areantibodies directed to Protein A of MRSA and MSSA and are usuallymonoclonal antibodies. In order to allow for adsorption of MRSA and/orMSSA, the specimen will be usually contacted for 5 to 120 minutes,preferably for 10 to 60 minutes with the antibodies.

[0018] After adsorption of MRSA and/or MSSA has taken place, theantibodies will be separated from the specimen. Depending on theemployed method of separation Anti-Protein A antibodies or modified e.g.biotinylated Anti-Protein A antibodies, both commercially available canbe used in the present invention. The specimen and the antibodies can beseparated by e.g. centrifugation, chromatography e.g. affinitychromatography or immobilisation of the antibodies or a combination ofthese methods. If immobilisation or affinity chromatography is used theantibodies can alternatively be immobilised or interact with theaffinity column first and might then be contacted with the specimen.

[0019] Preferably, immobilisation of Anti-Protein A antibodies is usedin the present invention to separate the antibodies having adsorbed MRSAand/or MSSA from the specimen. In particular a centrifugation step isadded before immobilising the antibodies. The antibodies can beimmobilised on e.g. paramagnetic beads. Streptavidin and biotin can beused as binding partners, e.g. by linking biotinylated Anti-Protein Aantibodies to streptavidin coated paramagnetic beads. Preferably,biotinylated Anti-Protein A antibodies and streptavidin coatedparamagnetic beads are used to immobilise the antibodies. Streptavidincoated paramagnetic beads are commercially available. Usually, theantibodies and the paramagnetic beads will be incubated to allow forbinding of the antibodies to the paramagnetic beads and then will beheld magnetically to separate from the specimen by washing theparamagnetic beads. The incubation period is usually 5 to 120 minutes,preferably 10 to 60 minutes. The period to held the paramagnetic beadsmagnetically is usually 1 to 60 minutes, preferably 2 to 20 minutes.

[0020] The antibodies can be resuspended during separation if necessary.For resuspending and washing e.g. phosphate buffered saline containinghuman serum albumin can be used.

Lysing of the Microorganisms Adsorbed to Release their DNA

[0021] MRSA and/or MSSA adsorbed to the antibodies can be lysed torelease their DNA by e.g. applying a detergent such as Tween, TritonX-100, an alkali such as NaOH, a protease such as proteinase K or achaotroph and/or shear forces. The lysis is usually carried out in abuffer like TE (Tris-HCl 10 mM, pH=8 and EDTA 1 mM) or in water(molecular grade purity) or in a buffer like phosphate-buffered saline(PBS) or in saline.

[0022] As chaotroph e.g. guanidinium isothiocyanate, urea or guanidinehydrochloride, preferably guanidinium isothiocyanate is used. In case achaotroph is used ATL buffer commercialized by Qiagen which containsguanidinium isothiocyanate is preferably used. In case a chaotroph isused, it is removed from the released DNA after lysis by e.g contactingthe released DNA to a DNA binding column such as Qlamp (Qiagen). The DNAeluted from the column can be then combined with the probes and/or theprimers or can be concentrated before.

[0023] Shear forces can be applied by e.g. adding beads and mixing thesolution. As beads, preferably glass beads, more preferably glass beadswith a diameter of 100-200 μm as commercialised by Schieritz andHauenstein AG (Switzerland) can be added. The use of glass beads isparticularly useful since it has been found that if the lysis is carriedout in PBS or in saline glass beads, particularly glass beads with adiameter of 100-200 μm as commercialised by Schieritz and Hauenstein AG(Switzerland) quantitatively bind the released DNA which efficientlyextracts DNA from other components. After elution of the DNA bound tothe glass beads using water or TE, the eluted DNA can be combineddirectly with the probes and/or the primers or can be concentratedbefore. Alternatively glass beads with bound DNA can be suspended in PBSor in saline and can be directly combined with the probes and/or theprimers.

[0024] In addition lysis applying shear forces can be carried out aswell in TE (Tris-HCl 10 mM, pH=8 and EDTA 1 mM) or in water (moleculargrade purity) using beads, preferably glass beads, more preferably glassbeads with a diameter of 100-200 μm as commercialised by Schieritz andHauenstein AG (Switzerland). This procedure allows for direct recoveryof the DNA in the liquid phase. The DNA in the liquid phase can becombined directly with the probes and/or the primers or can beconcentrated before.

[0025] The preferred way to carry out the lysis is to use shear forces.More preferably the lysis is carried out in a buffer like (PBS) or insaline, using glass beads, particularly glass beads with a diameter of100-200 μm as commercialised by Schieritz and Hauenstein AG(Switzerland).

Probes and/or Primers

[0026] The probes and/or primers of the present invention which arespecific for a target DNA sequence of the mecA gene of MRSA and/or MRSE,a target DNA sequence of MRSA other than a target DNA sequence of themecA gene and a target DNA sequence of MRSE other than a target DNAsequence of the mecA gene encompass the particular sequence of theparticular probe and/or primer, a sequence complementary therof, a parttherof having at least 10, preferably 15, more preferably 20 nucleotidesin length and a variant thereof. Variants encompass degeneratedsequences, deletions, insertions and substitutions at one or morenucleotide position, as long as each particular primer or probe is stillspecific for the particular target DNA sequence. Usually, probes andprimers are applied for each target DNA sequence. Preferably, a pair ofprimers such as a forward primer and a reverse primer, and a probe areapplied.

[0027] The terms “specific probes and/or primers” or “probes and/orprimers which are specific for a target DNA sequence” as used hereinrefer to probes, which have a homology of higher than 60%, preferablyhigher than 70%, more preferably higher than 80%, in particular higherthan 90% with the particular target DNA sequence to be amplified andprimers which have a homology of higher than 60%, preferably higher than70%, more preferably higher than 80%, in particular higher than 90% withthe DNA flanking the particular target DNA sequence to be amplified. Theprobes and primers, respectively, hybridize under stringent conditionse.g. at a temperature of between 55 and 65° C., preferably between 58and 62° C. and at a given salt concentration of between 3 and 10 mM,preferably between 4 and 6 mM, with the complement of the particulartarget DNA sequence to be amplified and with the DNA flanking thecomplement of the particular target DNA sequence to be amplified,respectively.

[0028] As a basis to select (i) probes and/or primers which are specificfor a target DNA sequence of the mecA gene of MRSA and/or MRSE, (ii)probes and/or primers which are specific for a target DNA sequence ofMRSA other than a target DNA sequence of the mecA gene and (iii) probesand/or primers which are specific for a target DNA sequence of MRSEother than a target DNA sequence of the mecA gene, database sequencessuch as GenBank or EMBL, fragments described in the literature orisolated fragments of the respective microorganism can be used.Preferably, database sequences such as GenBank or EMBL, more preferablyGenBank is used. In particular, probes and/or primers which are specificfor a target DNA sequence of the mecA gene of MRSA and/or MRSE areselected from the mecA gene sequence comprised by sequence number Y14051(Genbank), (ii) probes and/or primers which are specific for a targetDNA sequence of MRSA other than a target DNA sequence of the mecA geneare selected from the femA gene sequence of MRSA comprised by sequencenumber X17688 (Genbank) and (iii) probes and/or primers which arespecific for a target DNA sequence of MRSE other than a target DNAsequence of the mecA gene gene are selected from the femA gene sequenceof MRSE comprised by sequence number U23713 (Genbank).

[0029] Probes and/or primers which are specific for a target DNAsequence of MRSA other than a target DNA sequence of the mecA gene canbe as well selected from database sequences such as GenBank or EMBL,fragments described in the literature or isolated fragments of therespective microorganism which comprise target DNA sequences of MSSAprovided that these target DNA sequences are as well harboured by MRSA.

[0030] Probes and/or primers which are specific for a target DNAsequence of MRSE other than a target DNA sequence of the mecA gene canbe as well selected from database sequences such as GenBank or EMBL,fragments described in the literature or isolated fragments of therespective microorganism which comprise target DNA sequences of MSSEprovided that these target DNA sequences are as well harboured by MRSE.

[0031] Usually, it will be sufficient to select one target DNA sequenceof the mecA gene of MRSA or MRSE to design the probes and the primerwhich are specific for a target DNA sequence of the mecA gene of MRSAand/or MRSE since the mecA gene is between 95% and 100% usually higherthan 97% homologous between MRSA and MRSE. Preferably, the selectedtarget DNA sequence of the mecA gene is from MRSA.

[0032] As target DNA sequences gene of MRSA and MRSE which are nothomologous every DNA sequence other than a target DNA sequence of themecA gene which is harboured by 100% of MRSA and 100% of MRSE,respectively, can be used provided that the homology between both targetDNA sequences is lower than 90%, preferably lower than 85%, mostpreferably lower than 80% and that both sequences do not crosshybridizeunder the stringent conditions described above. Usually target DNAsequences of a gene such as the femA gene of MRSA (femA-SA) and the femAgene of MRSE (femA-SE) or other target DNA sequences of genes of MRSAand MRSE which are not related to each other are used as target DNAsequences. Preferably, target DNA sequences of the femA gene of MRSA(femA-SA) and of the femA gene of MRSE (femA-SE) are used.

[0033] The probes and/or primers which are specific for a target DNAsequence of the mecA gene of MRSA and/or MRSE, a target DNA sequence ofMRSA other than a target DNA sequence of the mecA gene and a target DNAsequence of MRSE other than a target DNA sequence of the mecA gene areusually designed to generate an amplification product of approximately100+/−50 nucleotides. The sequence of the primers and probes can beobtained by using standard programs and primer analysis software such as“Primer Express” (Perkin Elmer) and can be synthesised using standardmethods such as an automated DNA synthesizer

[0034] Primers which are specific for a target DNA sequence of the mecAgene of MRSA and/or MRSE usually consist of 13-30, preferably 20-26nucleotides in length. Probes which are specific for a target DNAsequence of the mecA gene of MRSA and/or MRSE usually consist of 15-40,preferably 25-35, nucleotides in length.

[0035] Primers which are specific for a target DNA sequence of MRSAother than a target DNA sequence of the mecA gene and for a target DNAsequence of MRSE other than a target DNA sequence of the mecA geneusually consist of 13-30, preferably 20-26 nucleotides in length and areusually selected in order to contain >3 mismatches to discriminatetarget DNA sequence of MRSA other than a target DNA sequence of the mecAgene from the target DNA sequence of MRSE other than a target DNAsequence of the mecA gene. Probes which are specific for a target DNAsequence of MRSA other than a target DNA sequence of the mecA gene andfor a target DNA sequence of MRSE other than a target DNA sequence ofthe mecA gene usually consist of 15-40, preferably 25-35, nucleotides inlength and are usually selected in order to contain >3 mismatches todiscriminate target DNA sequence of MRSA other than a target DNAsequence of the mecA gene from the target DNA sequence of MRSE otherthan a target DNA sequence of the mecA gene.

[0036] All primers and probes can be selected to be compatible with eachother as well as with the other sets of primers and probes in order toavoid self-hybridisation or hybridisation between different primersand/or probes, loop-formation and false priming site. After selectionthe primers and probes can be tested in pairs to assess theirsensitivity and specificity and can be combined in differentconcentrations of usually 50-900 nM, to define optimal reactionconditions during amplification.

[0037] Preferred probes and/or primers specific for a target DNAsequence of the mecA gene of MRSA and/or MRSE are selected from theprimers comprising the sequences SEQ ID No. 1 and 2 and the probecomprising the sequence SEQ ID No. 3, a sequence complementary therof, apart therof having at least 10, preferably 15, more preferably 20nucleotides in length and a variant thereof. Particular preferred probesand/or primers specific for a target DNA sequence of the mecA gene ofMRSA and/or MRSE are the primers comprising the sequences SEQ ID No. 1and 2 and the probe comprising the sequence SEQ ID No. 3.

[0038] Preferred probes and/or primers specific for a target DNAsequence of MRSA other than a target DNA sequence of the mecA gene areprobes and/or primers, a sequence complementary therof, a part therofhaving at least 10, preferably 15, more preferably 20 nucleotides inlength and a variant thereof which are specific for a target DNAsequence of the femA gene of MRSA.

[0039] Particular preferred probes and/or primers specific for a targetDNA sequence of the femA gene of MRSA are selected from the primerscomprising the sequences SEQ ID No. 4 and 5 and the probe comprising thesequence SEQ ID No. 6, and the primers comprising the sequences SEQ IDNo. 10 and 11 and the probe comprising the sequence SEQ ID No. 12, asequence complementary therof, a part therof having at least 10,preferably 15, more preferably 20 nucleotides in length and a variantthereof. Most preferred are the primers comprising the sequences SEQ IDNo. 4 and 5 and the probe comprising the sequence SEQ ID No. 6 and theprimers comprising the sequences SEQ ID No. 10 and 11 and the probecomprising the sequence SEQ ID No. 12, in particular the primerscomprising the sequences SEQ ID No. 10 and 11 and the probe comprisingthe sequence SEQ ID No. 12.

[0040] Preferred probes and/or primers specific for a target DNAsequence of MRSE other than a target DNA sequence of the mecA gene areprobes and/or primers, a sequence complementary therof, a part therofhaving at least 10, preferably 15, more preferably 20 nucleotides inlength and a variant thereof which are specific for a target DNAsequence of the femA gene of MRSE.

[0041] Particular preferred probes and/or primers specific for a targetDNA sequence of the femA gene of MRSE are selected from the primerscomprising the sequences SEQ ID No. 7 and 8 and the probe comprising thesequence SEQ ID No. 9 and the primers comprising the sequences SEQ IDNo. 13 and 14 and the probe comprising the sequence SEQ ID No. 15, asequence complementary therof, a part therof having at least 10,preferably 15, more preferably 20 nucleotides in length and a variantthereof. Most preferred are the primers comprising the sequences SEQ IDNo. 7 and 8 and the probe comprising the sequence SEQ ID No. 9 and theprimers comprising the sequences SEQ ID No. 13 and 14 and the probecomprising the sequence SEQ ID No. 15, in particular the primerscomprising the sequences SEQ ID No. 13 and 14 and the probe comprisingthe sequence SEQ ID No. 15.

Amplification

[0042] Conditions which permit amplification of said target DNAsequences are well known to the person skilled in the art. Usually, anamplification method such as polymerase chain reaction (PCR), nested PCRor multiplex PCR, ligase chain reaction, nucleic acid sequence-basedamplification (NASBA), self-sustained sequence replication (3SR), stranddisplacement amplification (SDA), branched DNA signal amplification(bDNA), transcription-mediated amplification (TMA) or cycling probetechnology (CPT) can be used. Preferably, multiplex PCR is used. In amultiplex PCR the aforementioned probes and/or primers are used incombination in a single PCR reaction together with the released DNA.

[0043] In case a PCR method is used as amplification method e.g. a DNApolymerase such as Taq DNA polymerase (Perkin Elmer) or AmpliTaq (PEApplied Biosystems), Hot GoldStar Taq polymerase (Eurogentec), AmpliTaqGold polymerase (PE Applied Biosystems), SureStart Taq (Stratagene) orPlatinum Taq (Gibco) can be used. Preferably, Hot GoldStar Taq DNApolymerase is used. The treatment to denature into a single-stranded DNAis usually a thermal treatment. Usual PCR conditions are e.g. thermalcycling conditions like incubation for 0.5-5 min. at 40°-60°, preferably1.5-2.5 min. at 45°-55°, followed by incubation for 5-15 min. at90°-97°, preferably 8-12 min. at 95°, followed by incubation for 5-30sec. at 90°-97°, preferably 10-20 sec. at 95°, followed by incubationfor 0.1-5 min. at 50°-70°, preferably 0.5-2 min. at 55°-65°, whereby thelast two incubation steps will be repeated up to 100 times preferably 50times. A further denaturation step can be added as first incubation stepin case a DNA polymerase is used.

[0044] Usually a commercially available reaction buffer is added to thereleased DNA combined with the probes and/or primers before incubation.The probes and/or primers are usually added in excess to the releasedDNA. Usual ratios of probe and primers for each target DNA sequence arebetween 1/3 and 2/1. The ratio of one primer over the other for eachtarget DNA sequence are usually between 1/3 and 3/1.

[0045] The presence and amount of the amplified target DNA sequences canbe detected by e.g. a method in which the size of the amplified targetDNA sequences is confirmed by electrophoresis, by hybridizing theamplified target DNA sequences with a labelled probe having a sequencecomplementary to the sequence of the target amplified or by real-timePCR, wherein the amplified target DNA sequences are detected duringamplification e.g. by measuring fluorescence during amplification usinga variety of fluorescent dyes emitting at different wavelengths.Real-time PCR systems such as TaqMan® (Perkin Elmer), ABI Prism SequenceDetection Systems (Applied Biosystems), SmartCycler® (Cepheid), I-Cycler(BioRad), LightCycler (Roche), R.A.P.I.D. (Idaho Technologies), DNAEngine Opticon (MJ Research), RotorGene (Corbett Research), and MX400(Stratagene) are the preferred methods. Particulary preferred are theABI Prism Sequence Detection Systems, the TaqMan® and the SmartCycler®system. Most preferred are the ABI Prism Sequence Detection Systems.Different fluorescent dyes and non-fluorescent quenchers depending onreal-time PCR the system used can be coupled with e.g. each of the saidprobes prior to amplification e.g. a fluorescent dye such as FAM(6-carboxyfluorescein), HEX(6-carboxy-2′,4,4′,5′,7,7′-hexachlorofluorescein), TET(6-carboxy-4,7,2′,7′-tetrachloro-fluorescein), JOE(6-carboxy-4′,5′-dichloro-2′,7′-dimethoxy fluorescein) or Texas-Red® canbe coupled at the 5′-end of the probe and a fluorescent quencher such asTAMRA (6-carboxy-N,N,N′,N′-tetramethylrhodamine) or a non-fluorescentquencher such as DABCYL (4-((4-(dimethylamino)phenyl)azo)benzoic acid)can be coupled at the 3′-end of the probe. In case real-time PCR isused, usually the cycle threshold (Ct) of the amplified target DNAsequences is detected whereby the difference of the Cts obtained for thetarget DNA sequence of the mecA gene of MRSA and/or MRSE and obtainedfor the target DNA sequence of MRSE other than the mecA gene indicatesthe presence and amount of MRSA.

[0046] The Ct value among triplicates for each DNA target sequence isusually used to determine the presence and amount of MRSA.

[0047] The preferred conditions which indicate the presence and amountof MRSA are when all following requirements are met:

[0048] 1) Smallest Ct for target DNA sequence of the mecA gene of MRSAand/or MRSE<40

[0049]2) Smallest Ct for target DNA sequence of MRSA other than the mecAgene<40

[0050]3) Difference between Ct for target DNA sequence of the mecA geneof MRSA and/or MRSE and Ct for target DNA sequence of MRSE other thanthe mecA gene (Ct target DNA sequence of the mecA gene of MRSA and/orMRSE−Ct target DNA sequence of MRSE other than the mecA gene)≦0

[0051] Preferably the difference between Ct for target DNA sequence ofthe mecA gene of MRSA and/or MRSE and the Ct for target DNA sequence ofMRSE other than the mecA gene is <0, more preferably between <0 and −1,in particular between −0.25 and −0.75.

[0052] MRSA is absent in all other conditions.

[0053] Usually, DNA from control strains are combined with the specificprobes and/or primers and are amplified under the same conditions as theDNA target sequences described supra in order to determine Ct for eachDNA target sequence. As control strains e.g. MRSA, MRSE, MSSE, MSSAstrains, particularly ATCC33591 (MRSA), ATCC25923 (MSSA), Clinicalisolates from nasal swabs (MRSE) and ATCC12228 (MSSE) can be used.

[0054] The term cycle threshold (Ct) used herein refers to theparticular cycle of amplification which allows for unequivocal detectionof the amplified product calculated as 6-12 times, preferably 8 times,the standard deviation of the background.

[0055] The Ct for the DNA target sequences is usually set by determiningthe background for each fluorescent dye as the average fluorescencevalue measured during 3-18 cycles, preferably 3-12 cycles. Afterbackground subtraction, the cycle threshold for the target DNA sequenceof MRSA other than a target DNA sequence of the mecA gene can becalculated as 6-12 times, preferably 8 times, the standard deviation ofthe background. A similar calculation can be performed for the targetDNA sequence of MRSE other than a target DNA sequence of the mecA gene.The threshold value for the target DNA sequence of MRSE other than atarget DNA sequence of the mecA gene can be compared with the Ctobtained from a MRSE control strain. The threshold value for the DNAtarget sequences of the mecA gene can be then adjusted so that the Ctfor the DNA target sequences of the mecA gene is equal to the Ct for thetarget DNA sequence of MRSA other than a target DNA sequence of the mecAgene. This adjustment is usually performed by comparing with Ct for thetarget DNA sequence of the mecA gene and the target DNA sequence of MRSAother than a target DNA sequence of the mecA gene obtained from a MRSAcontrol strain. Usually, 100 pg total DNA of the control strain is usedper amplification run to determine Ct for the target DNA sequence of themecA gene, for the target DNA sequence of MRSA and for the target DNAsequence of MRSE. Preferably an internal control is used by addingusually 1-100 pg, preferably 1 pg, of a target DNA sequence other than atarget DNA sequence of the mecA gene, preferably a target DNA sequenceof the femA gene of MSSA, from an MSSA strain such as strain ATCC25923(MSSA) to the reaction buffer before carrying out the amplificationmethod. In case an internal control is used the presence and amount ofMRSA is usually indicated when:

[0056] 1) Smallest Ct for target DNA sequence of the mecA gene of MRSAand/or MRSE<40

[0057] 2) Smallest Ct for target DNA sequence of MRSA other than themecA gene<40

[0058] 3) Difference between Ct for target DNA sequence of the mecA geneof MRSA and/or MRSE and Ct for target DNA sequence of MRSE other thanthe mecA gene (Ct target DNA sequence of the mecA gene of MRSA and/orMRSE−Ct target DNA sequence of MRSE other than the mecA gene) <0

[0059] MRSA is absent in all other conditions. In case an internalcontrol is used, the presence and amount of MRSA is preferably indicatedwhen the difference between Ct for target DNA sequence of the mecA geneof MRSA and/or MPSE and Ct for target DNA sequence of MRSE other thanthe mecA gene (Ct target DNA sequence of the mecA gene of MRSA and/orMRSE−Ct target DNA sequence of MRSE other than the mecA gene) is between<0 and −1, more preferably between −0.25 and −0.75. Consequently theaddition of an internal control allows to determine only two Cts, Ct fortarget DNA sequence of the mecA gene of MRSA and/or MRSE and Ct fortarget DNA sequence of MRSE other than the mecA gene, in order toindicate the presence and amount of MRSA. Ct for target DNA sequence ofMRSA other than the mecA gene can be additionally determined and shouldbe <40 for considering the run as valid.

[0060] Further objects of the present invention are primers and/orprobes specific for a target DNA sequence of the mecA gene of MRSAand/or MRSE selected from the primers comprising the sequences SEQ IDNo. 1 and 2 and the probe comprising the sequence SEQ ID No. 3, asequence complementary therof, a part therof having at least 10,preferably 15, more preferably 20 nucleotides in length and a variantthereof; primers and/or probes specific for a target DNA sequence of thefemA gene of MRSA selected from the primers comprising the sequences SEQID No. 4 and 5 and the probe comprising the sequence SEQ ID No. 6, andthe primers comprising the sequences SEQ ID No. 10 and 11 and the probecomprising the sequence SEQ ID No. 12, a sequence complementary therof,a part therof having at least 10, preferably 15, more preferably 20nucleotides in length and a variant thereof; and primers and/or probesspecific for a target DNA sequence of the femA gene of MRSE selectedfrom the primers comprising the sequences SEQ ID No. 7 and 8 and theprobe comprising the sequence SEQ ID No. 9 and the primers comprisingthe sequences SEQ ID No. 13 and 14 and the probe comprising the sequenceSEQ ID No. 15, a sequence complementary therof, a part therof having atleast 10, preferably 15, more preferably 20 nucleotides in length and avariant thereof.

[0061] A further object of the present invention is a diagnostic kit forthe detection of methicillin-resistant Staphylococcus aureus in aspecimen comprising any suitable combination of the nucleic acids havingthe nucleotide sequences of any one of SEQ ID No. 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14 and 15 a sequence complementary therof; a parttherofhaving at least 10, preferably 15, more preferably 20 nucleotidesin length and a variant thereof comprising in addition theaforementioned Anti-Protein A antibodies. Preferably, the kit comprisesin addition to the aforementioned Anti-Protein A antibodies glass beads.

EXAMPLES Example 1

[0062] Selection and Synthesis of Primers and Probes

[0063] Target DNA sequences of the mecA gene of MRSA, of the femA geneof MRSA and of the femA gene of MRSE were selected from Genbank sequencedatabase.

[0064] The Genbank sequences of the femA gene of MRSE (femA-SE, fromGenbank sequence U23713) and of the femA gene of MRSA (femA-SA, fromGenbank sequence X17688) have been aligned using the “Clustal” alignmentmethod (Baylor College of Medicine) and the regions showing the poorestlevel of homology were detected. The research of primers and probes,restricted to those partial sequences has been done using “PrimerExpress” (Perkin Elmer software developed for Taqman® applications).Probes and primers were designed in the aim to generate short PCRfragments (approximately 100+/−50 nucleotides). Probes and primersspecific for target DNA sequences of femA-SA and of femA-SE weredesigned to consist of nucleotides containing at least >3 mismatches todiscriminate between the sequence of femA-SA and femA-SE.

[0065] Pure DNA preparations of the control strains ATCC25923 (MSSA) andATCC12228 (MSSE) allowed to test the specificity of the selectedprimers/probes. No cross reaction was recorded in the Taqman®amplification assay, using the described probes and/or primers. Thesequences SEQ ID No. 4, 5 and 6 amplified the region of nucleotide 651to 788 of femA-SA. The sequences SEQ ID No. 7, 8 and 9 amplified theregion of nucleotide 1403 to 1547 of femA-SE. The sequences SEQ ID No.10, 11 and 12 amplified the region of nucleotide 1600 to 1694 offemA-SA. The sequences SEQ ID No. 13, 14 and 15 amplified the region ofnucleotide 1315 to 1414 of femA-SE.

[0066] The amplified region of the mecA gene was 321 to 419 (mecA, fromGenbank sequence Y14051). Based on the known sequences of the mecA gene,this region is highly conserved for Staphylococcus aureus (Y14051) andStaphylococcus epidermidis (Genbank sequence X52592).

[0067] All nucleotides have been synthesized and fluorescently labelledby Eurogentec (Seraing, Belgium) using Expedite™ automated synthesizer.The purification technique was based on an internally developed(Eurogentec) chromatography system, leading to equivalent or betterperformance than obtained using HPLC. All nucleotides (OliGold™) weredelivered lyophilised, were deprotected and purified by reverse phasechromatography. The absence of salts, ammonium and acid residues wasalso checked. All reagents used for oligonucleotide synthesis werequality controlled as described in the Glen Research catalogue. The dyesused FAM, TET, HEX JOE or Texas Red® ((bound to the 5′ end of the probe)and the quencher TAMRA or DABCYL (bound to the 3′ end) are coupled usingthe appropriate phosphoramidite or succinimidyl ester derivatives (FAM:6-carboxyfluorescein, TAMRA: 6-carboxy-N,N,N′,N′-tetramethylrhodamine,HEX: 6-carboxy-2′,4,4′,5′,7,7′-hexachloro-fluorescein, TET:6-carboxy-4,7,2′,7′-tetrachloro-fluorescein, JOE:6-carboxy-4′,5′-dichloro-2′,7′-dimethoxy fluorescein, DABCYL:(4-((4-(dimethylamino)phenyl)azo)ben-zoic acid, or Texas Red®).

[0068] Primers and probes were tested to be mutually compatible witheach other as well as with the other sets of primers and probes(multiplexing) in order to avoid self-hybridisation or hybridisationbetween different primers and/or probes, loop-formation and falsepriming site. Primers and probes were then tested in pairs to assesstheir sensitivity and specificity. They were then combined in differentconcentrations (50-900 nM) to define optimal reaction conditions.

Example 2

[0069] Preconditioning of the Samples

[0070] Samples consisting in nasal/inguinal swabs were collected andpossibly pooled for each patient by suspending them into CS medium(Brain-Heart Infusion with colistin and 2.5% NaCl). A preliminary studyshowed that this bacterial medium enriched in salt, bactericidal andnutritive agents did not influence the following steps and performedequally well as saline (0.9% NaCl). These samples (1 ml) were thensupplemented by 50 μl of 20% human serum albumin (injectable grade fromthe Swiss Red Cross). The preconditioning of the samples required about5 min.

[0071] Enrichment of the Sample

[0072] 1.5 μl of a mouse biotinylated monoclonal antibody raised againststaphylococcal protein A (Sigma) was then added to all samples, followedby 45-minutes incubation on a rotary shaker at room temperatureconditions. Samples were then centrifuged for 10 minutes at 5000 g. Thesupernatant was removed before adding 200 μl of phosphate bufferedsaline containing 1% human serum albumin (PBS-HSA). Samples were mixedwith 10 μl streptavidin-coated magnetic beads (ø=1 μm, Merck) andincubated on the rotary shaker for 45 minutes, in ambient conditions.Samples were then transferred into a 96 well plate and kept on amagnetic holder for 5 minutes (Milian). The supernatant was removed andthe paramagnetic beads were rinsed twice in PBS-HSA. The enrichment ofthe sample required about 95 min.

[0073] Bacterial Lysis and Release of DNA

[0074] The paramagnetic beads were then suspended in the 96 well platein 400 μl of chaotropic buffer (ATL buffer from the kit QIamp, Qiagen)and transferred into clean eppendorf tubes. Hundred milligrams of glassbeads (ø=100-200 μm, Schieritz and Hauenstein AG, CH-4144 Arlesheim)were added and bacteria were lysed using a mixing apparatus (Mixer Mill,Qiagen) with 2 homogenisation cycles of 45 seconds each. After 10minutes centrifugation at 5000 g, the liquid phase was placed into cleantubes and mixed with 200 μl of room-temperature pure ethanol. DNA wasimmobilized onto QIamp columns (Qiagen), rinsed as described by themanufacturer and eluted into a volume of 100 μl of pure water. Theextracts were then dried in an evaporator (UniVapo 100H, UniEquip) for 1hour and resuspended in 20 μl of pure water. The bacterial lysis andrelease of DNA required about 30 min.

[0075] Nucleic Acid Detection by Real-time PCR and Analysis

[0076] For the robustness of the analysis, each sample containing thereleased DNA was analysed in triplicate. Detection and analysis requiredabout 150 min. TaqMan™ system (Perkin Elmer) together with the HotGoldStar Taq DNA polymerase (Eurogentec) were used for amplification ofthe samples.

[0077] A. Amplified Targets TABLE 1 A B Forward Reverse C primer primerProbe 1 mecA CATTGATCGCAAC TGGTCTTTCTGCA TGTGGAAGTTAGA GTTCAATTTTTCCTGGA TTGGGATCATAGC GTCAT 2 femA-SA TGCCTTTACAGAT AGTAAGTAAGCAATCATTTCACGCAA AGCATGCCA GCTGCAATGACC ACTGTTGGCCACT ATG 3 femA-SECAACTCGATGCAA GAACCGCATAGCT TACTACGCTGGTG ATCAGCAA CCCTGC GAACTTCAAATCGTTATCG

[0078] Forward primer A1 corresponds to SEQ ID No. 1. Reverse primer B1corresponds to SEQ ID No. 2. Probe C1 corresponds to SEQ ID No. 3.Forward primer A2 corresponds to SEQ ID No. 4. Reverse primer B2corresponds to SEQ ID No. 5. Probe C2 corresponds to SEQ ID No. 6.Forward primer A3 corresponds to SEQ ID No. 7. Reverse primer B3corresponds to SEQ ID No. 8. Probe C3 corresponds to SEQ ID No. 9.

[0079] The final volume of the PCR mixture was 25 μl and contained thefollowing compounds:

[0080] Reaction buffer 2×: 12.5 μl (commercialized by Eurogentec)

[0081] A1: 75 nM, B1: 75 nM and C1: 100 nM

[0082] A2: 100 nM, B2: 100 nM and C2: 100 nM

[0083] A3: 100 nM, B3: 100 nM and C3: 100 nM

[0084] 6 μl of the sample and the amplification run was immediatelyperformed.

[0085] Fluorescent dyes linked to the probes were the following:

[0086] C1 (mecA) 5′-HEX and 3′-TAMRA

[0087] C2 (femA-SA) 5′-FAM and 3′-TAMRA

[0088] C3 (femA-SE) 5′-TET and 3′-TAMRA

[0089] B. Amplification Conditions:

[0090] The assay has been developed according to Applied Biosystemsspecifications, except that 50 cycles were used to increase thesensitivity (without loss of specificity). incubation 1 →  2 min. at 50°C. incubation 2 → 10 min. at 95° C. incubation 3 → 15 sec. at 95° C.incubation 4 →  1 min. at 60° C.

[0091] C. Analysis

[0092] After visual inspection of the data obtained, the background foreach fluorescent reporter was determined as the average fluorescencevalue measured during cycles 3-12. After background subtraction, thethreshold value for femA-SA was calculated as 6-12 times (preferably 8times) the standard deviation of the background. A similar calculationwas performed for femA-SE. Ct for femA-SE was determined using 100 pgDNA/well from control MRSE strain (isolate from a nasal swab) under thesame conditions as described in A and B. The threshold value for mecAwas then adjusted so that Ct for mecA=Ct for femA-SA. This determinationwas performed using 100 pg DNA/well from control MRSA strain (ATCC33591)under the same conditions as described in A and B. From screenshotsobtained for femA-SA, mecA and femA-SE the smallest Ct among triplicatesfor each DNA target sequence was determined. MRSA was present when allfollowing conditions were met:

[0093] 1) Smallest Ct for mecA<40

[0094] 2) Smallest Ct for femA-SA<40

[0095] 3) Ct for mecA−Ct for femA-SE≦0

[0096] MRSA was absent in all other conditions.

Example 3

[0097] Alternative Method for Bacterial Lysis and Release of DNA

[0098] Bacterial lysis and release of DNA of Example 1 was alternativelycarried out as follows:

[0099] a) Bacterial lysis was performed using glass beads in 0.9% sodiumchloride (NaCl) solution. After lysis, the supernatant was discarded andthe beads suspended in 200 to 400 μl of pure water. After vigorousagitation, eluted DNA (now in the liquid phase) was concentrated byevaporation, solubilized in 20 μl of water and used directly in theTaqman® amplification assay.

[0100] b) After bacterial lysis in the presence of 0.9% NaCl, the beadswere rinsed twice with the same solution, then suspended with 100 μl ofthe saline solution. 6 μl aliquots of the beads suspension were directlyintroduced in the Taqman® amplification assay.

[0101] c) Bacterial lysis was performed suspending glass beads in 200 to400 μl of pure water. After vigorous agitation, eluted DNA (now in theliquid phase) were concentrated by evaporation, solubilized in 20 μl ofwater and used directly in the Taqman® amplification assay.

Example 4

[0102] Evaluation of Sensitivity

[0103] Experiments using samples spiked with known amounts of MRSA andMRSE revealed a specificity of hundred percent and a sensitivity of 1genome copy. A large range of spiked amounts has been tested from 10 ngto 0.1-0.2 fg/reaction (1 genome copy equivalent to 1.65×10⁻¹⁵ g)

Example 5

[0104] Comparing Molecular Detection to Standard Culture-basedProcedures

[0105] A middle scale study (38 samples treated simultaneously)comparing molecular detection to standard culture-based proceduresperformed by the Laboratoire Central de Microbiologie, LCB, GenevaUniversity Hospital) allowed the detection of all positive culture cases(11/11). Two samples negative by culture were found positive using themolecular technique of example 1. For these 2 patients, the LCB foundthe presence of MRSA in another sampling site.

Example 6

[0106] A. Paramagnetic Enrichment of Mixed-samples Containing MRSA andMRSE

[0107] The samples were spiked with different ratios of MRSA and MRSE(from 1/1 to 1/1000). Samples were then either directly lysed (protocola) or enriched by the method described in example 1 using anti protein Aantibodies and paramagnetic beads (protocol b) and subject toamplification as described in example 2.

[0108] The results obtained showed that for the 1/1 ratio and despitethe additional steps in protocol b, the Cts were increased by 1-2 cycleswithout enrichment (protocol a). This implies an increased sensitivityof protocol b over protocol a. For the 1/5 ratio the Ct for femA-SA wereincreased from 2-2.5 cycles without enrichment (protocol a). Theincreased sensitivity was more string when the ratio MRSA/MRSEdecreased. For ratios lower than 1/5, no femA-SA signal was recordedusing protocol a in the tested range of spiked bacteria. Using protocolb, no significant signal for femA-SE was recorded even with ratios lowerthan 1/5, indicating an excellent enrichment even in the presence of alarge excess of MRSE.

[0109] B. Detection of MRSA and MRSE in the Presence of Pseudomonasaeruginosa

[0110] This experiment was based on a commonly encountered situation inclinical diagnosis where patients become colonized by Pseudomonasaeruginosa. Whenever Pseudomonas aeruginosa was spiked to the ratios asindicated in A, no femA SA signal could be recorded unless using theprotocol b.

[0111] For all conditions using protocol b, the Ct of femA SA and CtmecA were almost identical and the interpretation was straightforward,underlying the absolute need of this step for accurate and sensitivedetection.

Example 7

[0112] Preconditioning and enrichment of the sample were carried out asdescribed in example 2, except that the sample was suspended in PBS andthen supplemented by 50 μl of 20% human serum albumin (injectable gradefrom the Swiss Red Cross). Bacterial lysis and release of DNA wascarried out as described in example 3a) except that the enriched samplewas resuspended in 50 μl of pure water before adding the glass beads.After shearing force disruption, the DNA contained in the liquid phase(10 μl) was directly added to the amplification mixture and subject tothe Taqman® amplification assay.

[0113] Nucleic Acid Detection by Real-time PCR and Analysis

[0114] For the robustness of the analysis, each sample containing thereleased DNA was analysed in triplicate. Detection and analysis requiredabout 150 min. ABI Prism Sequence Detection System cycler (AppliedBiosystems) together with the Hot GoldStar Taq DNA polymerase(Eurogentec) was used for amplification of the samples.

[0115] A. Amplified Targets TABLE 2 A B Forward Reverse C primer primerProbe 1 mecA CATTGATCGCAAC TGGTCTTTCTGCA TGTGGAAGTTAGA GTTCAATTTTTCCTGGA TTGGGATCATAGC GTCAT 4 femA-SA GCTGGTGGTACAT CGGTCAATGCCATTCATTTTGCCGGA CAAATGCA GATTTAATG AGTTATGCAGT GCA 5 femA-SE CCCATCTCTGCTGCCGCATAGCTCCC CGCTGGTGGAACT GCTTCTTT TGCAA TCAAATCGTTA TCG

[0116] Forward primer A1 corresponds to SEQ ID No. 1. Reverse primer B1corresponds to SEQ ID No.2. Probe C1 corresponds to SEQ ID No. 3.Forward primer A4 corresponds to SEQ ID No. 10. Reverse primer B4corresponds to SEQ ID No. 11. Probe C4 corresponds to SEQ ID No. 12.Forward primer A5 corresponds to SEQ ID No. 13. Reverse primer B5corresponds to SEQ ID No. 14. Probe C5 corresponds to SEQ ID No. 15.

[0117] The final volume of the PCR mixture was 25 μl and contained thefollowing compounds:

[0118] Reaction buffer 2×: 12.5 μl (commercialized by Eurogentec)

[0119] A1: 100 nM, B1: 100nM and C1: 75 nM

[0120] A4: 50 nM, B4: 50 nM and C4: 50 nM

[0121] A5: 400 nM, B5: 400 nM and C5: 100 nM

[0122] 10 μl of the sample and the amplification run was immediatelyperformed.

[0123] Fluorescent dyes linked to the probes were the following:

[0124] C1 (mecA) 5′-JOE and 3′-DABCYL

[0125] C4 (femA-SA) 5′-FAM and 3′-DABCYL

[0126] C5 (femA-SE) 5′-Texas Red® and 3′-DABCYL

[0127] B. Amplification Conditions:

[0128] The assay has been developed according to Applied Biosystemsspecifications, except that 50 cycles were used to increase thesensitivity (without loss of specificity). incubation 1 →  2 min. at 50°C. incubation 2 → 10 min. at 95° C. incubation 3 → 15 sec. at 95° C.incubation 4 →  1 min. at 60° C.

[0129] C. Analysis

[0130] After visual inspection of the data, the background for eachfluorescent reporter was determined as the average fluorescence valuemeasured during cycles 3-12. After background subtraction, the thresholdvalue for each fluorescent reporter was calculated as 6-12 timespreferably 8 times) the standard deviation of the background. A similarcalculation was performed for femA-SE. Ct for femA-SE was determinedusing 100 pg DNA/well from control MRSE strain (isolate from a nasalswab) under the same conditions as described in A and B. The thresholdvalue for mecA was then adjusted so that Ct for mecA =Ct for femA-SA.This determination was performed using 100 pg DNA/well from control MRSAstrain (ATCC33591) under the same conditions as described in A and B.From screenshots obtained for femA-SA, mecA and femA-SE the smallest Ctamong triplicates for each DNA target sequence was determined. MRSA waspresent when all following conditions were met:

[0131] 1) Smallest Ct for mecA<40

[0132] 2) Smallest Ct for femA-SA<40

[0133] 3) Ct for mecA−Ct for femA-SE<0

[0134] MRSA was absent in all other conditions.

Example 8

[0135] For comparison purpose, the same samples as used in example 7were analyzed in parallel using primers and probes of Example 2 sectionA (Table 1) as well as primers and probes of Example 7 section A (Table2).

[0136] Results using primers and probes of Example 2: Ct femA-SA Ct mecACt femA-SE MRSA 31.5 31.5 50 MSSA 32 50 50 MRSE 50 30 30 MSSE 50 50 28.6

[0137] Results using primers and probes of Example 7: Ct femA-SA Ct mecACt femA-SE MRSA 27 27 50 MSSA 27.5 50 50 MRSE 50 26 26 MSSE 50 50 26

[0138] Primers and probes used in Example 7 provided similarinterpretation but offered increased sensitivity, i.e. smaller Ct valuesfor all three analyzed target genes.

Example 9

[0139] Example 9 was carried out as example 7 except that 1 pg ofgenomic DNA extracted from the MSSA strain ATCC25923 was added as aninternal control to the reaction buffer. After amplification, theanalysis was performed on the basis of the Ct obtained for mecA and theCt for femA-SE. Different spiked quantities ranging from 100 pg to 1 pgwere tested and confirmed the linearity of the determination within thisrange of concentrations, Cts for femA-SA were 24 and 30-31,respectively. Detection and analysis were performed on either the ABIPrism Sequence Detection Systems (Applied Biosystems) or the SmartCycler (Cepheid).

[0140] Both provided similar linear results

[0141] The presence and amount of MRSA was indicated when the differencebetween Ct for mecA−Ct for femA-SE<0. MRSA was absent in all otherconditions. Taking advantage of 1 pg of spiked genomic DNA from MSSA,the Ct for femA-SA was measured to confirm that the amplification workedproperly.

1 15 1 22 DNA Artificial Sequence Description of Artificial SequencePrimer specific for a target DNA sequence of the mecA gene of MRSAand/or MRSE 1 cattgatcgc aacgttcaat tt 22 2 21 DNA Artificial SequenceDescription of Artificial Sequence Primer specific for a target DNAsequence of the mecA gene of MRSA and/or MRSE 2 tggtctttct gcattcctgg a21 3 31 DNA Artificial Sequence Description of Artificial Sequence Probe specific for a target D NA sequence of the mecA gene of MRSA and/orMRSE 3 tgtggaagtt agattgggat catagcgtca t 31 4 22 DNA ArtificialSequence Description of Artificial Sequence Primer specific for a targetDNA sequence of the femA gene of MRSA 4 tgcctttaca gatagcatgc ca 22 5 25DNA Artificial Sequence Description of Artificial Sequence Primerspecific for a target DNA sequence of the femA gene of MRSA 5 agtaagtaagcaagctgcaa tgacc 25 6 29 DNA Artificial Sequence Description ofArtificial Sequence P robe specific for a target D NA sequence of thefemA gene of MRSA 6 tcatttcacg caaactgttg gccactatg 29 7 21 DNAArtificial Sequence Description of Artificial Sequence Primer specificfor a target DNA sequence of the femA gene of MRSE 7 caactcgatgcaaatcagca a 21 8 19 DNA Artificial Sequence Description of ArtificialSequence Primer specific for a target DNA sequence of the femA gene ofMRSE 8 gaaccgcata gctccctgc 19 9 32 DNA Artificial Sequence Descriptionof Artificial Sequence P robe specific for a target D NA sequence of thefemA gene of MRSE 9 tactacgctg gtggaacttc aaatcgttat cg 32 10 21 DNAArtificial Sequence Description of Artificial Sequence Primer specificfor a target DNA sequence of the femA gene of MRSA 10 gctggtggtacatcaaatgc a 21 11 22 DNA Artificial Sequence Description of ArtificialSequence Primer specific for a target DNA sequence of the femA gene ofMRSA 11 cggtcaatgc catgatttaa tg 22 12 27 DNA Artificial SequenceDescription of Artificial Sequence P robe specific for a target D NAsequence of the femA gene of MRSA 12 tcattttgcc ggaagttatg cagtgca 27 1321 DNA Artificial Sequence Description of Artificial Sequence Primerspecific for a target sequence of the femA gene of MRSE 13 cccatctctgctggcttctt t 21 14 18 DNA Artificial sequence Description of ArtificialSequence Primer specific for a target DNA sequence of the femA gene ofMRSE 14 ccgcatagct ccctgcaa 18 15 27 DNA Artificial Sequence Descriptionof Artificial Sequence P robe specific for a target D NA sequence of thefemA gene of MRSE 15 cgctggtgga acttcaaatc gttatcg 27

1. A method for the detection and quantification ofmethicillin-resistant Staphylococcus aureus from a specimen comprisinga) contacting the specimen with Anti-Protein A antibodies as to adsorbmethicillin-resistant Staphylococcus aureus and/or methicillin-sensitiveStaphyloccoccus aureus, b) separating said antibodies to whichmethicillin-resistant Staphylococcus aureus and/or methicillin-senstiveStaphylococcus aureus have been adsorbed from the specimen, c) lysingmethicillin-resistant Staphylococcus aureus and/or methicillin-sensitiveStaphylococcus aureus adsorbed to said antibodies so as to release theirDNA, d) combining the released DNA with (i) probes and/or primers whichare specific for a target DNA sequence of the mecA gene ofmethicillin-resistant Staphylococcus aureus and/or methicillin-resistantStaphylococcus epidermis, (ii) probes and/or primers which are specificfor a target DNA sequence of methicillin-resistant Staphylococcus aureusother than a target DNA sequence of the mecA gene and (iii) probesand/or primers which are specific for a target DNA sequence ofmethicillin-resistant Staphylococcus epidermis other than a target DNAsequence of the mecA gene, whereby the target DNA sequences ofmethicillin-resistant Staphylococcus aureus and of methicillin-resistantStaphylococcus epidermis are not homologous, e) subjecting the combinedreleased DNA and the specific probes and/or primers to conditions whichpermit amplification of said target DNA sequences, f) detecting thepresence and amount of the amplified target DNA sequences as anindication of the presence and amount of methicillin-resistantStaphylococcus aureus.
 2. A method of claim 1, whereinmethicillin-resistant Staphylococcus aureus and/or methicillin-sensitiveStaphylococcus aureus are lysed by applying shear forces.
 3. A method ofclaim 1, wherein the released DNA is combined with (i) probes andprimers which are specific for a target DNA sequence of the mecA gene ofmethicillin-resistant Staphylococcus aureus and/or ofmethicillin-resistant Staphylococcus epidermis, (ii) probes and primerswhich are specific for a target DNA sequence of methicillin-resistantStaphylococcus aureus other than a target DNA sequence of the mecA geneand (iii) probes and primers which are specific for a target DNAsequence of methicillin-resistant Staphylococcus epidermis other than atarget DNA sequence of the mecA gene.
 4. A method of claim 1, whereinthe probes and/or primers specific for a target DNA sequence of the mecAgene of methicillin-resistant Staphylococcus aureus and/or ofmethicillin-resistant Staphylococcus epidermis are selected from theprimers comprising the sequences SEQ ID NO. 1 and 2 and the probecomprising the sequence SEQ ID No. 3, a sequence complementary thereof,a part thereof having at least 10 nucleotides in length and a variantthereof.
 5. A method of claim 1, wherein the probes and/or primers whichare specific for a target DNA sequence of the methicillin-resistantStaphylococcus aureus other than a target DNA sequence of the mecA geneare probes and/or primers, a sequence complementary thereof, a partthereof having at least 10, preferably 15, more preferably 20nucleotides in length and a variant thereof which are specific fortarget DNA sequence of the femA gene of methicillin-resistantStaphylococcus aureus.
 6. A method of claim 1, wherein the probes and/orprimers which are specific for a target DNA sequence ofmethicillin-resistant Staphylococcus epidermis other than a target DNAsequence of the mecA gene are probes and/or primers, a sequencecomplementary thereof, a part thereof having at least 10, preferably 15,more preferably 20 nucleotides in length and a variant thereof which arespecific for a target DNA sequence of the femA gene ofmethicillin-resistant Staphylococcus epidermis.
 7. A method of claim 1,wherein the presence and the amount of the amplified target DNAsequences are detected by real-time PCR.
 8. A method of claim 7, whereinthe cycle treshold of the amplified target DNA sequences is detectedwhereby the difference of the cycle tresholds obtained for the targetDNA sequence of the mecA gene of MRSA and/or MRSE and obtained for thetarget DNA sequence of methicillin-resistant Staphylococcus epidermisother than the mecA gene indicates the presence and amount ofmethicillin-resistant Staphylococcus aureus.
 9. Primers and/or probesspecific for a target DNA sequence of the mecA gene of MRSA and/or MRSEselected from the primers comprising the sequence SEQ ID No. 1 and 2 andthe probe comprising the sequence SEQ ID No. 3, a sequence complementarythereof, a part thereof having at least 10, preferably 15, morepreferably 20 nucleotides in length and a variant thereof.
 10. Primersand/or probes specific for a target DNA sequence of the femA gene ofMRSA selected from the primers comprising the sequences SEQ ID No. 4 and5 and the probe comprising the sequence SEQ ID No. 6, and the primerscomprising the sequences SEQ ID No. 10 and 11 and the probe comprisingthe sequence SEQ ID No. 12, a sequence complementary thereof, a partthereof having at least 10, preferably 15, more preferably 20nucleotides in length and a variant thereof.
 11. Primers and/or probesspecific for a target DNA sequence of the femA gene of MRSE selectedfrom the primers comprising the sequences SEQ ID No. 7 and 8 and theprobe comprising the sequence SEQ ID No. 9 and the primers comprisingthe sequences SEQ ID No. 13 and 14 and the probe comprising the sequenceSEQ ID No. 15, a sequence complementary thereof, a part thereof havingat least 10, preferably 15, more preferably 20 nucleotides in length anda variant thereof.
 12. A diagnostic kit for the detection ofmethicillin-resistant Staphylococcus aureus in a specimen comprising anysuitable combination of the nucleic acids having the nucleotide sequenceof any one of SEQ ID No. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14and 15 a sequence complementary thereof, a part thereof having at least10 nucleotides in length and a variant thereof wherein the kit comprisesin addition Anti-Protein A antibodies.